Thiamine plays a critical role in the acid tolerance of Listeria monocytogenes

Understanding the molecular basis of acid tolerance in the food-borne pathogen Listeria monocytogenes is important as this property contributes to survival in the food-chain and enhances survival within infected hosts. The aim of this study was to identify genes contributing to acid tolerance in L. monocytogenes using transposon mutagenesis and subsequently to elucidate the physiological role of these genes in acid tolerance. One mutant harboring a Tn917 insertion in the thiT gene (formerly lmo1429), which encodes a thiamine (vitamin B1) uptake system, was found to be highly sensitive to acid. The acid-sensitive phenotype associated with loss of this gene was confirmed with an independently isolated mutant, from which the thiT gene was deleted (ΔthiT). Cells of both wild-type and ΔthiT mutant that were thiamine depleted were found to be significantly more acid sensitive than control cultures. Thiamine-depleted cultures failed to produce significant concentrations of acetoin, consistent with the known thiamine dependence of acetolactate synthase, an enzyme required for acetoin synthesis from pyruvate. As acetoin synthesis is a proton-consuming process, we suggest that the acid sensitivity observed in thiamine-depleted cultures may be owing to an inability to produce acetoin.

Critical success factors in urban brownfield regeneration: an analysis of ‘hardcore’ sites in Manchester and Osaka during the economic recession (2009–10)

Hardcore, or long-term derelict and vacant brownfield sites which are often contaminated, form a significant proportion of brownfield land in many cities, not only in the UK but also in other countries. The recent economic recession has placed the economic viability of such sites in jeopardy. This paper compares the approaches for bringing back hardcore brownfield sites into use in England and Japan by focusing on ten case studies in Manchester and Osaka, using an `agency'-based frame- work. The findings are set in the context of (i) national brownfield and related policy agendas; (ii) recent trends in land and property markets in both England and Japan; and (iii) city-level comparisons of brownfields in Manchester and Osaka. The research, which was conducted during 2009 ^ 10, suggests that hardcore brownfield sites have been badly affected by the recent recession in both Manchester and Osaka. Despite this, not only is there evidence that hardcore sites have been successfully regenerated in both cities, but also that the critical success factors (CSFs) operating in bringing sites back into use share a large degree of commonality. These CSFs include the presence of strong potential markets, seeing the recession as an opportunity, long-term vision, strong branding, strong partnerships, integrated development, and getting infrastructure into place. Finally, the paper outlines the policy implications of the research.

Mountain waves in two-layer sheared flows: Critical-level effects, wave reflection, and drag enhancement

Internal gravity waves generated in two-layer stratified shear flows over mountains are investigated here using linear theory and numerical simulations. The impact on the gravity wave drag of wind profiles with constant unidirectional or directional shear up to a certain height and zero shear above, with and without critical levels, is evaluated. This kind of wind profile, which is more realistic than the constant shear extending indefinitely assumed in many analytical studies, leads to important modifications in the drag behavior due to wave reflection at the shear discontinuity and wave filtering by critical levels. In inviscid, nonrotating, and hydrostatic conditions, linear theory predicts that the drag behaves asymmetrically for backward and forward shear flows. These differences primarily depend on the fraction of wavenumbers that pass through their critical level before they are reflected by the shear discontinuity. If this fraction is large, the drag variation is not too different from that predicted for an unbounded shear layer, while if it is small the differences are marked, with the drag being enhanced by a considerable factor at low Richardson numbers (Ri). The drag may be further enhanced by nonlinear processes, but its qualitative variation for relatively low Ri is essentially unchanged. However, nonlinear processes seem to interact constructively with shear, so that the drag for a noninfinite but relatively high Ri is considerably larger than the drag without any shear at all.

The emergence and use of diagramming in system dynamics: a critical account

The paper reviews the leading diagramming methods employed in system dynamics to communicate the contents of models. The main ideas and historical development of the field are first outlined. Two diagramming methods—causal loop diagrams (CLDs) and stock/flow diagrams (SFDs)—are then described and their advantages and limitations discussed. A set of broad research directions is then outlined. These concern: the abilities of different diagrams to communicate different ideas, the role that diagrams have in group model building, and the question of whether diagrams can be an adequate substitute for simulation modelling. The paper closes by suggesting that although diagrams alone are insufficient, they have many benefits. However, since these benefits have emerged only as ‘craft wisdom’, a more rigorous programme of research into the diagrams' respective attributes is called for.

Documents as 'critical incidents' in organisation to consumer communication

A diary study tracked the paper documents received by nine UK informants over one month. Informants gave simple ratings of individual documents’ attractiveness and the ease of understanding them; more detailed reactions to the documents were gathered through informant diaries and follow-up interviews. The detailed reactions extended beyond the feedback gathered through the rating task. Informants showed sensitivity to the content, language, design and circumstances of receipt of documents, with indications that they developed opinions of originating organizations based on their experience of using their documents. Documents that failed to provide all the information needed, that failed to make their intentions clear (or obscured their intentions) or that were perceived as miss-targeted received negative comment. Repeat experiences of receiving either well- or poorly-conceived documents strengthened informant reactions to individual originating organizations. The paper concludes with recommendations for steps document originators, writers and designers need to take to prepare documents that enhance organization to consumer communication. We recommend that organizations evaluate and act on consumers’ reactions to their documents, beyond user testing in document development or scorecard ratings in use.

Identification of critical residues in the active site of porcine membrane-bound aminopeptidase P

The membrane-bound form of mammalian aminopeptidase P (AP-P; EC 3.4. 11.9) is a mono-zinc-containing enzyme that lacks any of the typical metal binding motifs found in other zinc metalloproteases. To identify residues involved in metal binding and catalysis, sequence and structural information was used to align the sequence of porcine membrane-bound AP-P with other members of the peptidase clan MG, including Escherichia coli AP-P and methionyl aminopeptidases. Residues predicted to be critical for activity were mutated and the resultant proteins were expressed in COS-1 cells. Immunoelectrophoretic blot analysis was used to compare the levels of expression of the mutant proteins, and their ability to hydrolyze bradykinin and Gly-Pro-hydroxyPro was assessed. Asp449, Asp460, His523, Glu554, and Glu568 are predicted to serve as metal ion ligands in the active site, and mutagenesis of these residues resulted in fully glycosylated proteins that were catalytically inactive. Mutation of His429 and His532 also resulted in catalytically inactive proteins, and these residues, by analogy with E. coli AP-P, are likely to play a role in shuttling protons during catalysis. These studies indicate that mammalian membrane-bound AP-P has an active-site configuration similar to that of other members of the peptidase clan MG, which is compatible with either a dual metal ion model or a single metal ion in the active site. The latter model is consistent, however, with the known metal stoichiometry of both the membrane-bound and cytosolic forms of AP-P and with a recently proposed model for methionyl aminopeptidase.

Glycogen synthase kinase 3 (GSK3) in the heart: a point of integration in hypertrophic signalling and a therapeutic target? A critical analysis

Glycogen synthase kinase 3 (GSK3, of which there are two isoforms, GSK3alpha and GSK3beta) was originally characterized in the context of regulation of glycogen metabolism, though it is now known to regulate many other cellular processes. Phosphorylation of GSK3alpha(Ser21) and GSK3beta(Ser9) inhibits their activity. In the heart, emphasis has been placed particularly on GSK3beta, rather than GSK3alpha. Importantly, catalytically-active GSK3 generally restrains gene expression and, in the heart, catalytically-active GSK3 has been implicated in anti-hypertrophic signalling. Inhibition of GSK3 results in changes in the activities of transcription and translation factors in the heart and promotes hypertrophic responses, and it is generally assumed that signal transduction from hypertrophic stimuli to GSK3 passes primarily through protein kinase B/Akt (PKB/Akt). However, recent data suggest that the situation is far more complex. We review evidence pertaining to the role of GSK3 in the myocardium and discuss effects of genetic manipulation of GSK3 activity in vivo. We also discuss the signalling pathways potentially regulating GSK3 activity and propose that, depending on the stimulus, phosphorylation of GSK3 is independent of PKB/Akt. Potential GSK3 substrates studied in relation to myocardial hypertrophy include nuclear factors of activated T cells, beta-catenin, GATA4, myocardin, CREB, and eukaryotic initiation factor 2Bvarepsilon. These and other transcription factor substrates putatively important in the heart are considered. We discuss whether cardiac pathologies could be treated by therapeutic intervention at the GSK3 level but conclude that any intervention would be premature without greater understanding of the precise role of GSK3 in cardiac processes.

Where theory meets practice: a critical comparison of research into identifying letters and craft knowledge of type design

This paper introduces scientific research findings and accounts of skilled design judgement to: (i) develop an interdisciplinary account of what affects our identification of letters when reading; (ii) analyse the relationship between the approaches of psychologists and designers to explaining how we identify letters; (iii) propose ways in which collaboration may work to make psychological research more relevant to typographic practice. The topics reviewed are addressed within each discipline and cover the contribution of letters and words to reading; letter features; essential or structural forms; uniformity within font design; and letter spacing. Analysis of the literature identifies possible means of reconciling different perspectives, points out some anomalies in interpretation of findings, and proposes how designers may contribute to research planning and dissemination.